Non-virulent Porphyromonas gingivalis mutant

ABSTRACT

A non-virulent, recA defective mutant of  Porphyromonas gingivalis . The  Porphyromonas gingivalis  strain which is deposited at ATCC under accession number 202109. Also a method of decreasing the growth rate or reproduction rate of  Porphyromonas gingivalis  in a mammal comprising the step of administering to the mammal at least one dose of  Porphyromonas gingivalis  according to the present invention. Further, a method of preventing or treating a  Porphyromonas gingivalis  infection such as periodontitis in a mammal comprising the step of administering to the mammal at least one dose of  Porphyromonas gingivalis  according to the present invention. Further, a method of preventing or treating a  Porphyromonas gingivalis  infection such as periodonitis in a mammal comprising the step of administering to the mammal at least one dose of  Porphyromonas gingivalis  according to the present invention. Also, a pharmaceutical composition comprising a non-virulent, recA defective mutant of  Porphyromonas gingivalis.

CROSS-REFERENCE TO RELATED APPLICATION

The present Application is a national phase filing under 35 U.S.C. §371of PCT Application No. PCT/US99/18197, titled “Non-virulentPorphyromonas Gingivalis Mutant” and filed Aug. 11, 1999; which is acontinuation of U.S. patent application Ser. No. 09/133,089, titled“Non-Virulent Porphyromonas Gingivalis Mutant” and filed Aug. 12, 1998,now U.S. Pat. No. 6,254,863, issued Jul. 3, 2001; the contents of whichare incorporated in this disclosure by reference in their entirety.

BACKGROUND

Periodontitis is an inflammatory disease of the tissues surrounding theteeth characterized by loss of the periodontal ligament attachment andalveolar bone support of the tooth. Periodontitis affects more than 49million people in the United States and hundreds of millions of peopleworldwide and has been reported as a risk factor for cardiovasculardisease and pre-term delivery of low-birth-weight infants. The mostcommon cause of periodontitis is chronic Gram-negative bacterialinfections. Among the Gram-negative bacteria implicated as a cause ofperiodontitis, Porphyromonas gingivalis is the major component of theflora in over 90% of adult periodontitis lesions.

Besides being a major etiological agent in adult human periodontitis,Porphyromonas gingivalis also causes aspiration pneumonia andnecrotizing pneumonia, abscesses in brain, genitourinary tract and lung,as well as mediastinitis. By contrast, P. gingivalis is not normallyfound at healthy sites nor is it found in patients with gingivitis butwith no accompanying periodontitis.

The current therapy for periodontitis is directed toward identifying,removing and controlling the etiologic factors, and then correcting thedefects these pathogens have caused. These therapies include scaling androot planing, chemotherapy, periodontal surgery and periodic maintenancetherapy. However, these treatments are not entirely effective because,for example, the pathogens can become resistant to chemotherapeuticagents.

Several potential virulence factors have been identified which appear torelate to the pathogenicity of P. gingivalis in periodontitis. Thesefactors include fimbriae (adhesins), capsule (antiphagocytosis),lipopolysaccharide (bone resorption), proteases (specific andgeneralized tissue destruction) and a variety of toxic by-products(e.g., ammonia). Some of these factors have been purified andbiochemically characterized. However, the specific roles, interactions,relative importance and regulation of these factors remains to bedetermined.

Therefore, there remains a need for effective prevention and treatmentfor periodontitis. Further, there remains a need for a modified strainof P. gingivalis that can be used as a host genetic background todetermine the specific roles. interactions, relative importance andregulation of the potential virulence factors produced by wild-type P.gingivalis.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, there is provideda non-virulent, recA defective mutant of Porphyromonas gingivalis.According to another embodiment of the present invention, there isprovided a Porphyromonas gingivalis strain which is deposited at ATCCunder accession number 202109.

According to another embodiment of the present invention, there isprovided a pharmaceutical preparation comprising a mutant ofPorphyromonas gingivalis according to the present invention.

According to another embodiment of the present invention, there isprovided a method of decreasing the growth rate or reproduction rate ofPorphyromonas gingivalis in a mammal, such as a human. The methodcomprises the step of administering to the mammal at least one dose of anon-virulent, recA defective mutant of Porphyromonas gingivalis, such asat least one dose of a Porphyromonas gingivalis strain which isdeposited at ATCC under accession number 202109.

According to another embodiment of the present invention, there isprovided a method of preventing or treating a Porphyromonas gingivalisinfection such as periodontitis in a mammal, such as a human. The methodcomprises the step of administering to the mammal at least one dose ofPorphyromonas gingivalis according to the present invention.

The methods of the present invention can be performed by administeringto the mutant with the at least one dose of a non-virulent, recAdefective mutant of Porphyromonas gingivalis via a route selected fromthe group consisting of a subcutaneous route, an intravenous route andan intramuscular route, among other routes. In a preferred embodiment,the methods of the present invention include administering at least onedose of a non-virulent, recA defective mutant of Porphyromonasgingivalis, wherein the dose is between about a 1×10³ and 1×10⁷ bacteriaper kg of body weight of the mammal. More preferably, the dose isbetween about 1×10⁵ and 1×10⁶ bacteria per kg of body weight of themammal.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures where:

FIGS. 1 and 2 show the results of Southern blot analyses of allelicexchange mutants of P. gingivalis to confirm the presence of theermF-ermAM cassette in a predicted location;

FIG. 3 is a bar graph showing the results of an assay for argininespecific proteolytic activity of P. gingivalis FLL32, FLL33 and W83 inthe presence or absence of L-cysteine;

FIGS. 4 and 5 are bar graphs showing the results of an assay for thelocalization of arginine-specific proteolytic activity and forlysine-specific proteolytic activity, respectively, of P. gingivalisFLL32, FLL33 and W83 in the presence or absence of L-cysteine;

FIGS. 6 and 7 show the results of Northern blot analyses of prpRI andprtP protease genes, respectively, of P. gingivalis FLL32, FLL33 andW83;

FIG. 8 shows the results of an analysis by SDS-PAGE of the ability of P.gingivalis FLL32, FLL33 and W83 to degrade purified C3 complementprotein; and

FIG. 9 is a graph showing the results of accumulation of C3 fragments onthe bacterial surface of FLL32, FLL33 and W83.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the discovery of a non-virulent mutant ofPorphyromonas gingivalis. This mutant, designated FLL32, has been foundto convey protection against the wild-type Porphyromonas gingivalis W83in mammals when the mammal was immunized with the mutant strain FLL32.Further, FLL32 can be used as a host genetic background to determine thespecific roles, interactions, relative importance and regulation of thepotential virulence factors produced by wild-type P. gingivalis.

All publications mentioned in this document are incorporated byreference in their entirety.

A deposit of Porphyromonas gingivalis mutant strain FLL32 has been madeat the ATCC, Manassas, Va., US on Apr. 8, 1998, under the accessionnumber 202109. This deposit shall be viably maintained, replacing it ifit becomes non-viable, for a period of 30 years from the date of thedeposit, or for 5 years from the last date of request for a sample ofthe deposit, whichever is longer, and made available to the publicwithout restriction in accordance with the provisions of the law. TheCommissioner of Patents and Trademarks, upon request, shall have accessto the deposit.

In summary, the FLL32 strain was isolated during construction of amutant recA mutant of P. gingivalis W83 wild-type by allelic exchangemutagenesis. The FLL32 strain was recA⁻ and lacked black pigmentationand β-hemolytic activity on blood agar. Further, the FLL32 strain wasdeficient in proteolytic activity and significantly more sensitive to UVirradiation than the wild-type W83 strain.

The FLL32 strain exhibited substantially reduced virulence whenintroduced into mammals and protected those animals immunized with thatstrain against subsequent infection by the wild-type strain W83.Further, in Western blot experiments of whole cell extracts, uniqueimmunoreactive bands were found in FLL32 using sera from immunizedanimals. The FLL32 strain is the first nonvirulent recA strain of P.gingivalis shown to protect mammals against subsequent infection by thewild-type P. gingivalis.

Isolation and Characterization of P. gingivalis FLL32 Strain

Porphyromonas gingivalis FLL32 was isolated and characterized asfollows. First, the recA homolog gene was cloned from wild-type W83.Next, the recA homolog gene was sequenced. Then, an isogenic recA⁻mutant of P. gingivalis W83 designated FLL32 was constructed by allelicexchange mutagenesis and the presence of the defective recA DNA in theP. gingivalis FLL32 strain confirmed by Southern blot analyses.

Next, the phenotype and UV sensitivity of P. gingivalis FLL32 strain wasdetermined, as well as its arginine and lysine specific proteolyticactivity. Additionally, the amount of its mRNA transcript for the majorprotease genes was determined. Then, the amount of its C3 complementprotein degradation was determined and the amount of C3 accumulation onthe surface of the P. gingivalis FLL32 strain was determined. Finally,the virulence of the P. gingivalis FLL32 strain and protective effect ofimmunization by the FLL32 strain against subsequent challenge by thewild-type was examined.

(a) Cloning of the recA Homolog Gene from P. gingivalis W83

The recA homolog gene was cloned from P. gingivalis W83 as follows.First, degenerate oligonucleotide primers (Dybvig, K., et al.,“Degenerate oligonucleotide primers for enzymatic amplification of recAsequences from gram-positive bacteria and mycoplasmas.” J. Bacteriol.174, 2729-2732, 1992) were used in a polymerase chain reaction (PCR) toamplify a 320 bp fragment of the recA sequence of P. gingivalis W83.This PCR fragment was ³²P-labeled and used to screen a λ DASHrecombinant phage bank P. gingivalis W83 for the presence of hybridizingclones. Ten of 1×10³ phage clone plaques (1.0%) hybridized with theprobe.

The hybridizing phage plaques were then amplified and absorbed ontomaltose-grown E. coli cells. DNA from the phage clones was isolatedusing the Promega Lambda Wizard DNA Purification system (Promega,Corporation, Madison, Wis.). NotI-BamHI cleavage of purified DNA fromtwo of the recombinants, designated L2 and L10, revealed that the phageclones had different restriction fragment patterns. L2 contained an 8.0kb and a 6.5 kb fragment that were missing in L10, while L10 containedan 11 kb, a 5.8 kb and a 0.3 kb fragment that were missing in L2. BothL2 and L10 contained a similar 2.1 kb fragment. These data indicatedthat the L2 and L10 clones were independent clones and not siblings froma single cloning event.

The L10 clone was chosen for further study because it had the smallerfragment insert. Southern blot hybridization using the ³²P-labeled 0.3kb PCR fragment of the recA gene from the chromosome of W83 was used asa probe to identify the hybridizing fragment. The plasmid pUC19 was usedto subclone a 2.1 kb hybridizing BamHI fragment from L10. This clone wasdesignated pFLL26.

(b) Nucleotide Sequencing of the recA Homolog Gene

Both strands of the 2.1 kb hybridizing BamHI fragment from the L10 clonecarried on pFLL26 were sequenced and one 1.02 kb open reading framecorresponding to a 36 kDa protein was detected, GenBank Accession NumberU70054 (Fletcher et al., 1997). There was a start codon at base position774. A purine-rich sequence found in E. coli ribosome binding sites wasalso seen three bases upstream from the initiation site. Sequencesresembling procaryotic −10 and −35 promoter regions were detected atbase positions 749 and 729 respectively. The calculated G+C ratio forthe recA homolog gene was 50% which is close to the ratio of 46 to 48%previously reported for genomic P. gingivalis DNA (Shah and Collins,1988).

A comparison of the amino acid sequence of this gene with the NationalCenter for Biotechnology Information genetic sequence databank revealeda similarity of approximately 90, 86 and 82 percent to the RecA proteinsfrom Bacteroides fragilis, Prevotella ruminicola, GenBank AccessionNumber U21227, and Mycobacterium smegmatis, GenBank Accession NumberX99208, respectively. (Goodman and Woods, Molecular Analysis of theBacteroides fragilis recA Gene, Gene 94, pp. 77-82, 1990) Further,regions between amino acids 68 to 81 and 266 to 288 revealed conservedATP binding domains.

(c) Construction of a recA⁻Mutant in P. gingivalis W83

An isogenic recA⁻ mutant of P. gingivalis W83 was constructed by allelicexchange mutagenesis as follows. The nucleotide sequence of the clonedrecA fragment revealed a unique HincII restriction site at bp 435 of theopen reading frame (Fletcher, H. M. et al., “Nucleotide sequence ofPorphyromonas gingivalis W83 recA homolog and construction of arecA-deficient mutant.” Infect.Immun. 65, 4592-4597, 1997). To utilizethis site, a 1.8 kb EcoRI-PstI fragment containing the intact recA genewas subcloned into EcoRI-PstI cleaved pUC19. The resulting plasmid,pFLL23, was digested with HincII and ligated with the 2.1 kb ermF-ermAMcassette from pVA2298 to produce recombinant plasmid designated pFLL24.(See Fletcher, H. M., Schenkein, H. A., Morgan, R. M., Bailey, K. A.,Berry, C. R., and Macrina, F. L. (1995). Virulence of a mutant ofPorphyromonas gingivalis W83 that is defective in the prtH gene.Infect.Immun. 63, 1521-1528).

Then, the recombinant plasmid pFLL24 was used as donor DNA inelectroporation of P. gingivalis W83. Since the pFLL24 plasmid wasunable to replicate in P. gingivalis, Clindamycin resistant (Cc^(r))transformants could only arise as a result of an integration into thewild-type gene on the chromosome. Two double crossover events werepredicted between the regions flanking the erm marker and the wild-typerecA gene on the chromosome that would result in a replacement of asegment of the wild-type gene with a fragment conferring clindamycinresistance.

Following electroporation and plating on selective medium, 15 Cc^(r)colonies were detected after a 7 day incubation period. These colonieswere replica plated onto selective medium and exposed to UV light todetermine their sensitivity. Four UV sensitive colonies, designatedFLL32, FLL33, FLL34 and FLL35, were chosen from the unexposed plate forfurther study.

To confirm the presence of the ermF-ermAM cassette in the predictedlocation, that is interrupting the recA DNA, Southern blot analyses wereperformed on the total cellular DNA from P. gingivalis wild-type W83, asa control, and from the Cc^(r) transformants FLL32, FLL33, FLL34 andFLL35. Their DNA was cleaved with BamHI, electrophoresed through 0.7%agarose, bidirectionally transferred to nitrocellulose and probed with³²P labeled pFLL23 and pVA2198. If the DNA was digested with BamHI, apredicted 2.1 kb fragment would be seen. If the DNA was not digestedwith BamHI, a predicted 4.2 kb fragment would be seen. Since theermF-ermAM cassette is missing a BamHI site, any of the four Cc^(r)transformants with the ermF-ermAM cassette interrupting the recA DNAsequence should have shown a 4.2 kb fragment but not a 2.1 kb fragment.

Referring now to FIGS. 1 and 2, there are shown the results of theSouthern blot analyses of allelic exchange mutants of P. gingivalis toconfirm the presence of the ermF-ermAM cassette in the predictedlocation. As can be seen in FIG. 1, the predicted 2.1 kb fragment wasseen in the wild-type P. gingivalis W83, lane A, using the ³²P-labeledpFLL23 that carries the P. gingivalis recA homolog as a probe, indicatedthe presence of the recA DNA. In contrast, a 4.2 kb fragment was presentin each of the four Cc^(r) mutants of W83, lanes B-E, FLL32, FLL33,FLL34 and FLL35, respectively, and indicated the presence of the recADNA sequence interrupted by the ermF-ermAM cassette.

As can be seen in FIG. 2, using pVA2198, which carried the ermF-ermAMcassette as a probe, revealed an identical 4.2 kb hybridizing fragmentpresent in the four Cc^(r) mutants, lanes B-E, FLL32, FLL33, FLL34 andFLL35, respectively, but not in the wild-type W83, lane A, indicatingthe presence of the ermF-ermAM cassette only in the transformants. pUC19vector sequences used as a control did not hybridize with W83 or any ofthe four Cc^(r) mutants, FLL32, FLL33, FLL34 and FLL35 (data not shown).These data indicated that recombination had occurred in the four Cc^(r)mutants, FLL32, FLL33, FLL34 and FLL35, resulting in the wild-type recAgene being interrupted by the ermF-ermAM cassette in FLL32, FLL33, FLL34and FLL35.

(d) Phenotypic Characterization of P. gingivalis W83 recA Mutants

The phenotype of P. gingivalis W83 recA mutants were initiallycharacterized as follows. The recA mutants, FLL32, FLL33, FLL34 andFLL35, were plated on Brucella blood agar plates (Anaerobic Systems,Inc., San Jose, Calif.) to determine if any pleotropic effects wereassociated with inactivation of the recA gene. Two classes of mutantswere observed. The first class, a single colony FLL32, was unpigmentedand displayed significantly less β-hemolysis than the wild-type W83. Thesecond class contained three strains, FLL33, FLL34 and FLL35, all ofwhich displayed similar P-hemolytic activity and black pigmentation asthe wild- type W83. FLL32 and FLL33 were chosen for further study asrepresentatives of their respective classes. A generation time of 3hours was determined for W83 and of 3.5-4 hours for both FLL32 andFLL33.

(e) Determination of the UV Sensitivity of P. gingivalis W83 recAMutants

To confirm the loss of activity of the P. gingivalis RecA protein, therelative sensitivity of the wild-type and recA⁻ strains to UVirradiation was determined as follows. Wild-type W83 and recA⁻ mutantsFLL32 and FLL33 were exposed to 1000 μjoules of UV irradiation. Therewas an 80% survival of the wild-type W83 strain in contrast to the 18%survival for FLL32 and FLL33. When wild-type W83 and mutants FLL32 andFLL33 were exposed to 2000 μjoules of UV irradiation, there was 40%survival of the wild-type W83 cells compared to 0% survival for therecA⁻ mutants FLL32 and FLL33. These data indicated that the recA geneof P. gingivalis W83 plays an important role in repairing DNA damagecaused by UV irradiation and that both FLL32 and FLL33 were recAdefective.

(f) Determination of the Arginine and Lysine Specific ProteolyticActivity of FLL32, FLL33 and W83

The arginine specific proteolytic activity of P. gingivalis W83 recAmutants was determined by assaying whole cell preparations from each ofthe three strains of P. gingivalis, FLL32, FLL33 and W83 for proteolyticactivity using N-α-benzoyl-DL-arginine p-nitroanilide (BAPNA). Eachstrain of P. gingivalis was grown for 48 hours to late log phase (OD₆₀₀of 1.2) in 500 ml BHI broth supplemented with hemin and vitamin K. Thecells were then washed in PBS (pH 7.3) and resuspended to an OD₆₀₀ of0.3. 50 μl of the cell samples were incubated for 10 min at 37° C. in 50mM Tris-HCl (pH 7.0), and 1 mM α-N-benzoyl-arginine-DL-ρ-nitroanilide(BAPNA) in the presence or absence of 0.5 mM L-cysteine. The controlcontained buffer alone. Hydrolysis of BAPNA was monitored by the changeof absorbance at 410 nm.

Referring now to FIG. 3, there is shown a bar graph of the assayresults. As can be seen, the wild-type W83 (−Cys) showed moreproteolytic activity than FLL33 (−Cys), while FLL32 (−Cys) did not showsignificantly more proteolytic activity than the control. The activityfrom all three strains was enhanced in the presence of cysteine (+Cys)but the relative rates of proteolytic activity remained the same. Thereduction of proteolytic activity seen in FLL33 compared to W83 could berelated to the longer generation time for the recA⁻ strains compared tothe wild-type W83.

Localization of the arginine-specific proteolytic activity and forlysine-specific proteolytic activity in the recA⁻ strains was determinedas follows. First, extracellular proteolytic activity was tested.Ammonium sulfate was added to 500 ml of culture supernatant from cellsgrown to late log phase (OD₆₀₀ of 1.2) to 100% saturation. Theprecipitate was resuspended in 3 ml of PBS (pH 7.3), dialyzed againstthe same buffer, and then stored at −20° C.

Referring now to FIGS. 4 and 5, there are shown bar graphs showing theresults of an assay for the localization of arginine-specificproteolytic activity and for lysine-specific proteolytic activity. Ascan be seen, the FLL33 showed more extracellular arginine-specificproteolytic activity and more lysine-specific proteolytic activity thanthe wild-type W83 (−Cys). FLL32 did not show significantly morearginine-specific proteolytic activity or lysine-specific proteolyticactivity than the control. Further, the extracellular arginine-specificproteolytic activity of both W83 and FLL33, but not FLL32, was enhancedin the presence of cysteine (+Cys).

Next, intracellular proteolytic activity was tested. The cells from theabove experiment were washed in PBS (pH 7.4), and then resuspended inthe same buffer to a final volume of 10 ml. 1 ml aliquots weretransferred to microcentrifuge tubes containing 0.5 volume of 0.1 mmzirconium beads (Biospec Products, Inc. Bartlesville, Okla.), then lysedin a Mini-Bead Beater homogenizer (Biospec Products) for 3 min. Beadsand cellular debris were removed by centrifugation at 12,000×g for 5 minto obtain a clear lysate. Using 100 μg of protein per assay, similarintracellular arginine- and lysine-specific proteolytic activities wereobserved for the W83 and FLL33 strains, but there was no significantintracellular arginine- or lysine-specific proteolytic activities forFLL32 (data not shown).

(g) Comparison of the Presence and Amount of mRNA Transcript for theMajor Protease Genes in FLL32, FLL33 and W83

The loss of proteolytic activity in strain FLL32 could have resultedeither from a lack of transcription or translation of the gene, or froma lack of post-translational activation of the precursor product. Inorder to determine the cause of the loss of proteolytic activity inFLL32, the presence and amount of mRNA transcript for the major proteasegenes in FLL32, FLL33 and W83 was determined as follows.

First, total RNA was isolated using the Qiagen RNeasy Kit (Qiagen,Valencia, Calif.) from the wild-type W83 strain and from the FLL32 andFLL33 mutants grown to mid-log phase (OD₆₀₀ of 0.2). Uniqueoligonucleotide primers for prtP (as disclosed in Barkocy-Gallagher, G.A. et al., “Analysis of the prtP gene encoding porphypain, a cysteineproteinase of Porphyromonas gingivalis.” J.Bacteriol. 178, 2734-2741,1996), prpRI (Aduse Opoku, J. et al., “Characterization, geneticanalysis, and expression of a protease antigen (PrpRI) of Porphyromonasgingivalis W50.” Infect.Immun. 63, 4744-4754, 1995) and prtRII were usedin RT-PCR to amplify a 1 kb region of the transcripts. Amplifiedproducts of the predicted 1 kb size were observed for all three proteasegene transcript in all three strains (data not shown). Further, therewere no observed differences seen in the concentration of the amplifiedproduct between the genes of the three strains. Therefore, both FLL32and FLL33 strains produce the same mRNA transcripts for the majorprotease genes in the same amounts as the wild-type W83. As a control,recA intragenic primers amplified the expected 0.72 kb region only inthe wild-type W83 strain.

The presence of the mRNA transcripts for the prpRI and prtP proteases inall three P. gingivalis strains were further confirmed in Northern blotanalysis using an amplified intragenic region of each gene as a probe.Total RNA was extracted from each of the W83, FLL32 and FLL33 strainsgrown to mid-log phase (OD₆₀₀ of 0.2) using the Qiagen RNeasy midi kit(available from Qiagen, Valencia, Calif., according to themanufacturer's instructions). RNA samples of 1 μg were then separated byagarose gel electrophoresis and transferred to nitrocellulose filteraccording to the method of Sambrook et al. (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual.Second edition. (Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress)).

Referring now to FIGS. 6 and 7, there are shown the results of theNorthern blot analysis of prpRI and prtP protease genes from the threestrains of P. gingivalis W83, FLL33 and FLL32. FIG. 6 shows the resultsusing a ³²P-labeled specific intragenic region of prpRI as the probe.FIG. 7 shows the results using a ³²P-labeled specific intragenic regionof prpP as the probe. The size of the transcripts in kb are given in theleft margins. Lane A shows the results for the W83 wild-type strain,lane B shows the results for the FLL32 mutant strain and lane C showsthe results for the FLL33 mutant strain.

As can be seen in FIG. 6, the prpRI probe hybridized to 6.3 and 4.2 kbtranscripts. As can be seen in FIG. 7, the prtP probe hybridized to 6.6,4.3 and 3.2 kb transcripts. The 6.3 and 6.6 kb transcripts for the prpRIand prtP genes, FIGS. 6 and 7 respectively, are consistent with theknown size of those genes transcripts. The presence of the smallertranscripts could be degraded product or could be transcripts that shareregions of homology with the protease genes. These results confirm thepresence of the mRNA transcripts of the prpRI and prtP protease genes inall three strains of P. gingivalis, W83, FLL32 and FLL33.

(h) Determination of the C3 Complement Protein Degradation of FLL32,FLL33 and W83

The ability of P. gingivalis FLL32, FLL33 and W83 to degrade purified C3complement protein was determined as follows. 1 mg/ml of C3 wasincubated with increasing dilutions of each strain at 37° C. for 30minutes and the supernatant were analysed by SDS-PAGE with 10%separation gels and strained with comassie. The results are shown inFIG. 8 using C3 alone as a control (lane 1), 10⁹ W83 cells/ml (lane 2),5×10⁸ W83 cells/ml (lane 3), 10⁸ W83 cells/ml (lane 4), 10⁹ FLL32cells/ml (lane 5), 5×10⁸ FLL32 cells/ml (lane 6), 10⁸ FLL32 cells/ml(lane 7), 10⁹ FLL33 cells/ml (lane 8), 5×10⁸ FLL33 cells/ml (lane 9) and10⁸FLL33 cells/ml (lane 10).

As can be seen, the highest concentration of W83 tested (lane 2)completely degraded the α-chain of C3 with generation of C3b and somelower molecular mass fragments similar to C3c and C3d. The lowestbacterial concentration of W83 tested (lane 4) partially degraded theα-chain, causing both α and α′-chains to be visible. Similar resultswere observed for FLL33 (lanes 8-10). In contrast, the highestconcentration of FLL32 (lane 6) only minimally degraded C3 to C3b andlower molecular mass cleavage fragments. There was no degradation of C3at the lowest bacterial concentration tested for FLL32. Thus, FLL32 isless capable of degrading C3 than either the wild-type W83 or FLL33.

(i) Assessment of C3 Accumulation on FLL32, FLL33 and W83

Opsonization of P. gingivalis strains W83, FLL32 and FLL33 was assessedfor the accumulation of C3 fragments on the bacterial surface asfollows. 5×10⁸ cells/ml of each strain was incubated in pooled humanserum that was diluted 1:3 with Veronal-buffered saline (0.01 M Veronalbuffer, pH 7.5, containing 0.13 M NaCl) and that contained 2 μg/ml¹²⁵I-C3 for 35 minutes. The incubated bacterial samples were then washedand assessed for bound ¹²⁵I-C3 fragments by scintillation counting.Referring now to FIG. 9, it can be seen that W83 failed to accumulatesubstantial amounts of C3 by the end of the incubation period. Incontrast, FLL33 accumulated 3×10 molecules/bacterium and FLL32accumulated 6×10⁴ molecules/bacterium of ¹²⁵I-C3 fragments. Takentogether, these results suggest that FLL32 has an increased capacity tobe opsonized with C3 fragments compared to both W83 and FLL33.

EXAMPLE I Comparison of the Virulence Between FLL32, FLL33 and W83 in aMammal

A first comparison of the virulence between wild-type W83, mutant strainFLL32 and mutant strain FLL33 Porphyromonas gingivalis in a mammal wasmade as follows. Sixteen female Balb/c mice (8-10 weeks old, HarlanSprague Dawley Inc., Indianapolis Ind.) were divided into three groups,five in Group I, five in Group II and six in Group III. Each animalreceived a single challenge dose of 1×10¹⁰ bacteria P. gingivalis W83(Group I), FLL33 (Group II) or FLL32 (Group III) by subcutaneous, dorsalsurface injection, a dosage of approximately 2×10⁴ bacterial per kg bodyweight.

At 24 hours post-challenge, two of the five animals in Group I and oneof the five animals in the Group II had died and the remaining animalsin both Groups I and II appeared cachectic and hunched with ruffledhair. Although the animals did not display lesions at the dorsal surfacesite of injection (primary site), all had developed spreading,ulcerative abdominal skin lesions (secondary site). All of the remaininganimals in the Group I and three of the four remaining animals in theGroup II died by 48 hours post-challenge. The fifth animal in the GroupII died by the fourth day post-challenge.

In contrast, all six of the animals in Group III challenged with FLL32survived the 14 day post-challenge observation period. None of theanimals in Group III had any observable negative effects from thechallenge.

The data from these challenges were analyzed using Fisher's Exact Test.The analysis found no difference in the virulence between W83 and FLL33(p=1.000). However, the FLL32 strain had a statistical difference invirulence when compared to FLL33 (p=0.002) and W83 (p=0.002).

A second comparison of the virulence between wild-type W83, mutantstrain FLL33 and mutant strain FLL32 Porphyromonas gingivalis in amammal was made as follows. Seventeen mice Balb/c mice (8-10 weeks old,Harlan Sprague Dawley Inc., Indianapolis Ind.) were divided into threegroups, five in Group IV, six in Group V and six in Group VI. Eachanimal received a single challenge dose of 5×10⁹ bacteria P. gingivalisW83 (Group IV), FLL33 (Group V) or FLL32 (Group VI) by dorsalsubcutaneous surface injection.

At 24 hours post-challenge, one of five animals in Group IV had died andthe remaining four had developed ulcerated abdominal skin lesions. By 48hours post-challenge, three of the remaining animals in Group IV haddied. The lesions in the surviving fifth animal were resolving at day 14post-challenge.

At 24 hours post-challenge, one of six animals in Group V had died andthe remaining five had developed ulcerated abdominal lesions. By 48hours post-challenge, three of the five remaining animals in Group V haddied. One additional animal died by day 5 post-challenge. The lesions inthe surviving sixth animal were resolving at day 14 post-challenge.

In contrast, all six of the animals in Group VI challenged with FLL32survived the 14 day post-challenge observation period. None of theanimals in Group VI had any observable negative effects from thechallenge.

The results of these challenges were analyzed using Fisher's Exact Test.The analysis found no difference in the virulence between W83 and FLL33(p=0.727). However, the FLL32 strain had a statistical difference invirulence when compared to FLL33 (p=0.008) and W83 (p=0.015).

As can be appreciated from this Example, the inactivation of the recAgene in P. gingivalis FLL33 did not significantly affect the virulenceof P. gingivalis. However, the mutation in the FLL32 strainsignificantly affected the virulence of P. gingivalis.

EXAMPLE II Demonstration of the Protective Effect of Immunization withFLL32 Against Subsequent Challenge with Wild-Type W83 P. gingivalis

The protective effect of immunization of a mammal with FLL32 againstsubsequent challenge with wild-type W83 P. gingivalis was demonstratedas follows. Sixteen female Balb/c mice (8-10 weeks old, Harlan SpragueDawley Inc., Indianapolis Ind.) were subcutaneously immunized once perweek for 3 weeks with 1×10¹ bacteria of the mutant strain FLL32, adosage of 5×10⁵ bacteria per kg of body weight. Ten additional femaleBalb/c mice (8-10 weeks old, Harlan Sprague Dawley Inc., IndianapolisInd.) were subcutaneously immunized once per week for 3 weeks withsterile phosphate-buffered saline (PBS) as a control. All of the animalsimmunized with FLL32 and five of the ten animals immunized with PBS werethen challenged 2 weeks after the final immunization by subcutaneousinjection of a P. gingivalis W83 wild-type suspension containing 1×10¹⁰cells, a dosage of 5×10⁵ bacteria per kg of body weight. The remainingfive animals immunized with PBS were challenged 2 weeks after the finalimmunization by subcutaneous injection of PBS as a control.

By 24 hours post-challenge, one of the five control animals immunizedwith PBS and challenged with P. gingivalis W83 died and the other fouranimals had developed spreading infections with secondary site abdominalskin ulcerations and, in some, primary site ulcerations around the baseof the tail. All of these mice exhibited severe cachexia with ruffledhair, hunched bodies and weight loss; and all of these five controlanimals died by four days post-challenge.

In contrast, eight of sixteen animals immunized with P. gingivalis FLL32and challenged with P. gingivalis W83 displayed only minor secondaryskin site abdominal infections by 24 hours post-challenge but allrecovered and were alive at the end of the test period. Of the remainingeight animals immunized with P. gingivalis FLL32 and challenged with P.gingivalis W83, five had severe cachexia and died by three dayspost-challenge, two had moderate cachexia and developed secondaryulcerating abdominal lesions which began to heal at day 5 post-challengeand were alive at the end of the fourteen day experiment period, and thelast animal developed a secondary lesion which healed but then developedan additional secondary lesion and died at day 7 post-challenge.

All of the five animals immunized with PBS and challenged with PBSappeared normal throughout the fourteen day experiment period.

The results of these challenges were analyzed using Fisher's Exact Test.The analysis found that immunization with the FLL32 strain protected theanimals from a wild-type challenge (p=0.148), while those animals thatwere immunized with sterile phosphate-buffered saline were not protected(p=0.023).

At the end of the fourteen day experiment period, the ten survivinganimals from the group originally immunized with FLL32 and thenchallenged with W83, and the five animals immunized with PBS andchallenged with PBS were sacrificed and their sera were isolated toascertain the presence of anti-FLL32 antibodies. A 1:1000 dilution ofthe sera was tested by Western blot analysis for cross-reactivity towhole cell lysates of P. gingivalis W83, FLL32 and FLL33.

Animals immunized with FLL32 were positive for antibodies to each of theP. gingivalis whole cell lysates (data not shown). Immunoreactive bandswith molecular mass of 96, 82, 74, 55.2, 49.6, 38, 37, and 35 kDa wereobserved in the Western blot analyses of each of the whole cell lysatesof FLL32, FLL33 and W83. Immunoreactive bands with molecular mass of 44and 40 kDa were present in the Western blot analyses of each of thewhole cell lysates of FLL33 and W83 but were absent from the Westernblot analysis of the whole cell lysates of FLL32. Further,immunoreactive bands with molecular mass of 185, 170, 125, 71, 68, 63and 47 kDa were present in the Western blot analyses of whole celllysates of FLL32 but were absent in the Western blot analysis of each ofthe whole cell lysates of FLL33 and the W83 strain.

In contrast, sera from animals immunized with PBS and challenged withPBS were negative for antibodies to each of the P. gingivalis whole celllysates.

EXAMPLE III Method of Decreasing the Growth Rate or Reproduction Rate OfPorphyromonas gingivalis in a Mammal

According to one embodiment of the present invention, there is provideda method of decreasing the growth rate or reproduction rate ofPorphyromonas gingivalis in a mammal, such as a human. The methodcomprises the step of administering to the mammal at least one dose of anon-virulent, recA⁻ mutant of Porphyromonas gingivalis, such as FLL32.The dose can be administered, for example, by subcutaneous,intramuscular or intravenous injection. In a preferred embodiment, thedosage is between about 1×10³ and 1×10⁷ bacteria per kg of body weight.In a particularly preferred embodiment, the dosage is between about1×10⁵ and 1×10⁶ bacteria per kg of body weight.

Among the uses of decreasing the growth rate or reproduction rate ofPorphyromonas gingivalis in a mammal, such as a human, is the preventionor treatment of periodontitis, or other diseases or conditions caused inwhole or in part by Porphyromonas gingivalis, such as aspirationpneumonia and necrotizing pneumonia, abscesses in brain, genitourinarytract and lung, and mediastinitis

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the spirit and scope of the appended claims shouldnot be limited to the description of preferred embodiments containedherein.

I claim:
 1. A method of decreasing the growth rate or reproduction rateof wild-type Porphyromonas gingivalis in a mammal, the method comprisingadministering to the mammal at least one dose of a non-virulent, recAdefective mutant of Porphyromonas gingivalis which is deposited at ATCCunder accession number
 202109. 2. The method of claim 1, wherein themammals a human.
 3. The method of claim 1, wherein the administrationcomprises injecting the mammal with the at least one dose of thenon-virulent, recA defective mutant of Porphyromonas gingivalis via aroute selected from the group consisting of a subcutaneous route, anintravenous route and an intramuscular route.
 4. The method of claim 1,wherein the dose administered is between about 1×10³ and 1×10⁷ themutant of Porphyromonas gingivalis per kg of body weight of the mammal.